Fgfr-fc fusion protein and use thereof
a fusion protein and protein technology, applied in the field of biotechnology, can solve the problems of ineffective inhibition of tumor blood vessel growth over the long term, complicated dynamic process of angiogenesis regulation, and inability to effectively inhibit the growth of tumor blood vessel, etc., to achieve the effect of inhibiting angiogenesis and/or treating and/or preventing
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example 1
Construction of Recombinant Expression Plasmid for FGFR1-Fc Fusion Protein
[0132]The FGF receptor fragment is obtained from the amplification of the cDNA templet of FGF receptor through PCR, and IgG1 Fc fragment is obtained from the cDNA amplification of the human-derived IgG1 through PCR. A commercially available cDNA (PCR Ready First Strand cDNA, derived from human adult colon cancer tissue, BioChain) was used as the template for FGFR1 fragment. Total RNA was extracted from the blood of healthy human subjects using human blood RNA extraction kit (QIAGEN). According to the manufacturer's instruction of reverse transcription kit (Promega), RT-PCR was performed using M-MLV reverse transcriptase (Promega) so that RNA was reversely transcripted to cDNA which was used as the template for IgG1 Fc fragment. RT-PCR was performed according to the manufacturer's instruction of reverse transcription kit, which has the following steps: Oligo dT, dNTP, total RNA and DEPC H2O were mixed homogeneo...
example 2
Transient Expression and Quantification of the Fusion Proteins
[0136]The DNA of individual fusion protein plasmid was purified using MAX Plasmid Purification Kit (Qiagen). The concentration of the plasmid DNA was determined by UV spectrophotometry. 1 μg recombinant plasmid and 6 μL liposome (FuGENE 6 Transfection Reagent, Roche) were homogeneously mixed into 100 μL fresh IMDM culture broth (GIBCO); after standing for 15 min, the mixture was added to the CHO cells (ATCC) cultured overnight after inoculation at a cell density of 3×105 / mL into a 6-well plate; the mixture was cultured at 37° C. in an incubator supplemented with 5% CO2 for 48 h with a cell complete culture broth (IMDM medium containing 10% FBS, 1% HT and 1% glutamine, all supplied by GIBCO); subsequently, the supernatant was collected and determined for the relative content of the fusion protein using human IgG ELISA kit for protein quantification (BETHYL). The relative content of the fusion protein expressed and secreted...
example 3
Binding of the Fusion Proteins
[0137]The binding capacity of 19#, 13#, 22#, 23#, 26#, 29# and 8# fusion protein constructed above to FGF-2 was detected by ELISA.
[0138]Initially, a 96-well ELISA plate (IMMULON Company) was coated by 100 μL solution containing 50 ng / mL FGF-2 (R&D Systems) as well as containing 100 ng / mL heparin (Sigma Company) and 50 ng / mL FGF-2. Subsequently, the plate was washed by 300 μL PBST washing solution for 5 times before each coated well was blocked by 200 μL freshly prepared blocking working solution (KPL Company) (blocking stock solution:PBS=1:19) and incubated at 37° C. for 1 h. After washed in 300 μL PBST washing solution for 5 times, 100 μL solutions of various fusion proteins (dissolve in PBS, pH=7.2, concentration of 20 ng / ml) were added and incubated at 37° C. for 2 h. After washed in 300 μL PBST washing solution for 5 times, 100 μL secondary antibody to human IgG Fc-HRP (BETHYL Company) diluted with PBS in a ratio of 1:10000 was added and incubated a...
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